Preferentially enhancing anti-cancer isothiocyanates over glucosinolates in broccoli sprouts: How NaCl and salicylic acid affect their formation

Accepted Manuscript Preferentially enhancing anti-cancer isothiocyanates over glucosinolates in broccoli sprouts: How NaCl and salicylic acid affect their formation Azadeh Esfandiari, Ali Saei, Marian J. McKenzie, Adam Matich, Mesbah Babalar, Donald A. Hunter PII:

S0981-9428(17)30123-7

DOI:

10.1016/j.plaphy.2017.04.003

Reference:

PLAPHY 4849

To appear in:

Plant Physiology and Biochemistry

Received Date: 27 November 2016 Revised Date:

26 March 2017

Accepted Date: 5 April 2017

Please cite this article as: A. Esfandiari, A. Saei, M.J. McKenzie, A. Matich, M. Babalar, D.A. Hunter, Preferentially enhancing anti-cancer isothiocyanates over glucosinolates in broccoli sprouts: How NaCl and salicylic acid affect their formation, Plant Physiology et Biochemistry (2017), doi: 10.1016/ j.plaphy.2017.04.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Preferentially enhancing anti-cancer isothiocyanates over glucosinolates in broccoli sprouts: how NaCl and salicylic acid affect their formation Azadeh Esfandiari1,2*, Ali Saei1, Marian J. McKenzie1, Adam Matich1, Mesbah Babalar2, Donald

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A. Hunter1* 1. The New Zealand Institute for Plant & Food Research Limited, Private Bag 11600, Palmerston North 4442, New Zealand

of Horticulture, College of Agriculture & Natural Resources, University of Tehran, Karaj, Iran *[email protected] , [email protected]

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2. Department

ABSTRACT: Broccoli (Brassica oleracea L. var. italica) sprouts contain glucosinolates (GLs)

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that when hydrolysed yield health promoting isothiocyanates such as sulforaphane (SF). SF content can be increased by salt (NaCl) stress, although high salt concentrations negatively impact plant growth. Salicylic acid (SA) treatments can attenuate the negative effects of salt on growth. To test whether sprout isothiocyanate content could be elevated without sprout growth being compromised, broccoli seed were germinated and grown for seven days in salt (0, 80 and 160 mM) alone and in combination with 100 µM SA. Increasing concentrations of salt lowered

Gluconapin,

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transcript accumulation of GL biosynthetic genes which was reflected in lowered content of 4-methoxyglucobrassicin

and

neoglucobrassicin

glucosinolates.

Other

glucosinolates such as glucoraphanin did not alter significantly. Salt (160 mM) increased

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transcript abundance of the GL hydrolytic gene MYROSINASE (BoMYO) and its cofactor EPITHIOSPECIFIER MODIFIER1 (BoESM1) whose encoded product directs MYROSINASE to produce isothiocyanate rather than nitrile forms. SF content was increased 6-fold by the 160

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mM salt treatment, but the salt treatment reduced percentage seed germination, slowed seed germination, and reduced the elongation of the sprout hypocotyls. This growth inhibition was prevented if 100 µM SA was included with the salt treatment. These findings suggest that the increase in SF production by salt occurs in part because of increased transcript abundance of genes in the hydrolytic pathway and that this transcriptional enhancement occurs independently of salts more general negative impact on sprout growth.

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Keywords: Brassica oleracea, glucosinolates, sulforaphane, 2-phenethyl isothiocyanate, indol3-carbinol

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Abbreviations: GL, glucosinolate; SA, salicylic acid; NaCl, sodium chloride; CYP, Cytochrome; PAPS, 3`-phosphoadenosine-5`-phosphosulfate; BoMYO, broccoli myrosinase gene; BoESP, broccoli epithiospecifier gene; BoESM, broccoli epithiospecifier modifier protein gene; P5CS, delta-1-pyrroline-5-carboxylate; PR-1, pathogenesis related protein 1; GR, glucoraphanin; GI, glucoiberverin; GE, glucoerucin; GS, glucoalyssin; GB, 4hydroxyglucobrassicin; Mgb, 4-methoxyglucobrassicin; Ngb, neoglucobrassicin; GN, gluconasturtiin; PEITC, 2-phenethyl isothiocyanate; SF, sulforaphane; SFN, sulforaphane nitrile.

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1. Introduction Epidemiological and animal studies have shown that consumption of brassica vegetables,

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particularly broccoli, can prevent coronary heart disease and cancer (Royston and Tollefsbol, 2015). The compounds in the Brassicaceae responsible for these health benefits are the hydrolysis products of glucosinolates (GLs) (Kadir et al., 2015). GLs (thioglucosides) are watersoluble sulphur-rich secondary metabolites that are synthesised from amino acids and glucose via aliphatic and indolic pathways. Both pathways have enzymes in common and reciprocally inhibit

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each other (Gigolashvili et al., 2009). In the aliphatic pathway GLs are mainly derived from chain-elongated methionine, which is first converted to an aldoxime by CYTOCHROME P450

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79F1 (CYP79F1) and then to a nitrile oxide by CYP83A1. This nitrile oxide is then modified by a number of enzymes, shared with the indolic pathway, to eventually produce the aliphatic core GL structure. In the indolic pathway CYP79B2 converts tryptophan to indole-3-acetaldoxime and CYP83B1 converts tryptophan to indole-3-acetonitrile oxide, which is then modified further by a number of enzymatic steps to the indole core GL structure (Gao et al., 2014). The sulfur required for the final step of core GL biosynthesis is provided by the co-substrate 3`(PAPS)

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phosphoadenosine-5`-phosphosulfate

produced

by

ADENOSINE-5'-

PHOSPHOSULFATE (APS) KINASE (APSK) of the sulphur assimilation pathway (Sønderby et al., 2010). The importance of PAPS for both the indolic and aliphatic pathways is evidenced by a knockout mutant of APS kinase having substantially reduced GL content (Mugford et al., 2009).

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Research on Arabidopsis has uncovered specific R2R3-type MYB transcription factors as activators of the core GL pathways. Overexpression of MYB28, MYB29 and MYB76 increases the

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amount of methionine-derived aliphatic GLs, whereas overexpression of MYB51, MYB34, and MYB122 elevates quantities of tryptophan-derived indolic GLs (Gigolashvili et al., 2009). GL hydrolysis occurs in damaged tissues because the compounds become exposed to the endogenous enzyme myrosinase. Myrosinase converts GLs to isothiocyanates such as sulforaphane (SF), 2-phenylethylisothiocyanate (PEITC) and other hydrolysis products like indole-3-carbinol and neoascorbigen (Bones and Rossiter, 1996). It is the hydrolysis products of GLs that confer health benefits to humans (Ku et al., 2013b). These chemicals induce human quinine reductase, a cancer phase 2 detoxification enzyme that eliminates carcinogens from the human body (Boddupalli, 2012). The type of isothiocyanate produced by myrosinase depends on

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which indolic or aliphatic GL is present in the tissue, the tissue pH, concentration of Fe2+, and the type of cofactor of myrosinase present, i.e. whether it is epithiospecifier protein (ESP) or epithiospecifier modifier protein (ESM) (Bones and Rossiter, 1996; Nordstrom et al., 2013). It is the relative concentrations of the ESP and ESM cofactors that determines whether nitriles or

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isothiocyanates are produced (Burow et al., 2008). Under non-stressed conditions the ESP cofactor directs myrosinase to produce nitriles, which are inactive from an anti-cancer perspective (Bones and Rossiter, 1996; Ku et al., 2014). However, in certain circumstances, the ESM cofactor is produced and interferes with the ESP cofactor, thus leading to isothiocyanate

cancer compound (Matusheski and Jeffery, 2001).

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production (Burow et al., 2008). Of the reported isothiocyanates, SF is the most potent anti-

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The quantity of GLs in tissues is affected by NaCl, UV radiation, light, nitrogen, sulfur, glucose, fructose, selenium, SA, jasmonic acid, and pathogen attack (del Carmen Martínez-Ballesta et al., 2013; Singh et al., 2014). Researchers have found that NaCl treatments alter GL content in broccoli and radish, with the concentration of NaCl used being critical for whether GLs accumulate or decline. López-Berenguer et al. (2008) increased the total GL content of 20-dayold broccoli seedlings by growing them in 40 mM NaCl and decreased them by growing them in

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80 mM NaCl. Guo et al. (2013a) found that 4-day-old broccoli sprouts had higher SF and GR content and elevated myrosinase activity when they were treated with 160 mM NaCl. Guo et al. (2013b) further showed that the GL content in broccoli sprouts was decreased by the lower 20, 40 and 60 mM NaCl treatments. The GL content of seven-day-old radish sprouts was also

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differentially affected by the NaCl concentration in which they were grown, with lower NaCl concentrations (10 and 50 mM) suppressing total GL content and higher salt concentrations (100

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mM) increasing the total GL content of the sprouts (Guo et al., 2013b). The increasing salt concentration also significantly inhibited germination of the radish seed (Yuan et al., 2010). Therefore the mechanism of GL regulation under salinity is complex and still not completely understood (del Carmen Martínez-Ballesta et al., 2013) and the deleterious effects of NaCl on seed germination and plant growth is a substantial problem (Jayakannan et al., 2015). Salicylic acid (SA) is an endogenous hormone that has an important role in defence against biotic and abiotic stresses (Jayakannan et al., 2015). The hormone has been found to lessen the negative effects of salt stress in tissues (Hayat et al., 2010). For example, SA inhibited the NaClmediated reduction in growth of Brassica juncea seedlings, in part by enhancing the

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concentrations of the antioxidant enzymes catalase, peroxidase and superoxide dismutase (Yusuf et al., 2008).

The hormone also increased the amounts of proline and improved the

photosynthetic capacity of the seedlings by increasing chlorophyll content and carbonic anhydrase activity (Yusuf et al., 2008). Similarly, treatment with SA enhanced the growth of

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wheat plants under water-deficit stress (Singh and Usha, 2003), and maize, barley, tomato and Arabidopsis plants under NaCl stress (El-Tayeb, 2005; Gharbi et al., 2015; Horváth et al., 2015; Khodary, 2004). Young broccoli sprouts are thought to provide greater health benefits than head tissue, because they have much higher concentrations of the SF precursor, glucoraphanin (Fahey

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et al., 1997).

In this study, we investigated how continuous exposure of broccoli sprouts to various

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concentrations of NaCl affected sprout growth, transcription and metabolite content of the GL biosynthesis and hydrolysis pathways. We further determined how all of these salt-altered parameters were affected when the sprouts were co-treated with SA, a hormone previously

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documented to alleviate salinity-induced retardation of plant growth.

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2. Materials and methods 2.1. Plant Materials and growth conditions Seed of a commercial quality sprouting line broccoli (Brassica oleracea L. var. italica ‘Early

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Green’) were obtained from King Seeds (Katikati, New Zealand). All seeds were surface sterilised according to Chaudhary et al. (2014). For the GL/isothiocyanate measurements and transcript abundance analysis, c. 5 g of seed were sown onto two layers of filter paper in a sterile 15-cm diameter polypropylene tub. The tubs were transferred to a controlled environment

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chamber with a 16-h-light, 8-h-dark cycle and an air temperatures of 22 and 18°C, respectively (Avila et al., 2013). Treatments including SA (0 and 100 µM) and NaCl (0, 80 and 160 mM) and

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combinations were applied by pipetting 5 mL of each solution onto filter paper and then the liquid in each pot was renewed every two days with the solution according to the method of Ozdener and Kutbay (2008) For the GL/isothiocyanate measurements and transcript abundance analysis, the sprouts in each replicate were collected carefully so as not to damage and induce myrosinase activity, frozen in liquid nitrogen and stored at −80°C. For measurements of growth parameters, 60 seed were germinated in a polypropylene tub with the same treatments. The

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numbers of germinated seed were counted three times every day at the same time (c. 0900 h, 1300 h, 1600 h) for each of the three replicates. At day seven, 20 whole sprouts were removed from each replicate tub for aerial part (hypocotyl and cotyledons) length and fresh weight measurements.

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2.2. RNA extraction and quantitative real time PCR Total RNA was isolated from sprout tissue (ground under liquid nitrogen and stored at -80°C)

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using the ZR Plant RNA MiniPrep™ RNA extraction kit (www.zymoresearch.com). The RNA was treated with DNase I (Roche) and cDNA synthesized from 1 µg of the DNase-free RNA with SuperScript III (Invitrogen) and random hexamer primers (Roche). After synthesis, the cDNA was diluted 20-fold for use in quantitative RT-PCR (qRT-PCR). qRT-PCR was performed in 10-µL reactions using a SsoAdvanced Universal SYBR® Green Supermix PCR labelling kit (Bio-Rad) and a Rotor-Gene 3000 Real Time PCR machine (Corbett Research). Primers were designed using PrimerQuest software (www.idtdna.com). Target genes and primer sequences are listed in Table 1. PCR was performed on three biological replicates, each assayed in triplicate. Transcript abundance was normalised to abundance of BoACT1 using the method of

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(Livak and Schmittgen, 2001) and expressed relative to that of the control (non-treated sprouts at day 7).

2.3. Determination of glucosinolate concentration

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Sprout tissue samples (c. 5 g) that had been stored at -80°C were ground with dry ice and boiled in 10 times (w/v) 80% ethanol in Schott bottles for 5 min and held at −20°C overnight. The ethanolic extract was filtered through glass wool and the filtrate concentrated in a Savant™ Speed Vac™ concentrator at 40°C to a volume of 5 mL. A 1.8-mL aliquot of each extract was

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then transferred to a 2-mL Eppendorf tube and centrifuged at 10,000 rpm (13,000 x g) for 20 minutes at 4°C, and 30 µL of the supernatant diluted 50-fold (up to 1.5 mL) in a 2-mL vial. The

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external standards used for quantification (using [M-H]- current peak areas) were glucoerucin (4(methylthio)butylglucosinolate; Cfm Oskar Tropitzsch, Marktredwitz, Germany) for the aliphatic and phenolic glucosinolates, and glucobrassicin (indol-3-ylmethylglucosinolate; Cfm Oskar Tropitzsch) for the indolic glucosinolates. The external standard and the 1.5-mL sprout extracts were spiked with an internal standard (epicatechin, Sigma-Aldrich) to enable correction for sample volumes and for possible changes in Liquid Chromatogaphy-Mass Spectrometry (LC-

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MS) system responses (Maldini et al., 2012; Mullaney et al., 2013; Tian et al., 2005). 2.4. Liquid Chromatography-Mass Spectrometry Analysis Separations were performed on a Dionex Ultimate® 3000 Rapid Separation LC with identification and quantification by a micrOTOF-Q II mass spectrometer (Bruker Daltonics,

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Germany) fitted with an electrospray source operating in negative mode. Sample injections (1 mL), were made onto a dual column system: Two Zorbax™ SB-C18 HD 2.1 x 150 mm, 1.8 mm

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(Agilent) analytical columns maintained at 60°C and with a flow of 350 µL min-1. Solvent A was 0.2:99.8 HCO2H:H2O, and Solvent B was CH3CN. The solvent ramp was 10:90 (Solvent A: Solvent B) from injection to 0.5 min, followed by a linear gradient to 35:65 (0.5–15 min), to 50:50 (15–18 min), to 100:0 (18–22 min), hold at 100:0 (22–32 min). The micrOTOF-Q II source parameters were: temp. 200°C; drying N2 flow 8 L min-1; nebuliser N2 4 bar (400 kPa), endplate offset -500 V, capillary voltage +3500 V; mass range 100–1500 Da at 2 scans s-1. Postacquisition internal mass calibration used sodium formate clusters with the sodium formate delivered by a syringe pump at the start of each analysis. Mass spectral data were processed using Compass DataAnalysis (Bruker Daltonics) (Matich et al., 2015).

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2.5. Analysis of glucosinolate hydrolysis products Powdered sprout tissue samples (c. 10 g) that had been stored at -80°C were suspended in 2 times (w/v) water at 21°C for 4 h in sealed 100-mL Schott bottles covered in foil to prevent exposure to light. The extraction time of 4 h was chosen as it resulted in the best yield of

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isothiocyanates (Table 2). Three times (w/v) Et2O was then added and the solution left overnight at 1°C. The ether extracts were filtered through glass wool, dried with anhydrous MgSO4, and reduced in volume to 1.5 mL in a RapidVap N2 evaporator (Labconco, Kansas City)(Matich et al., 2015). The external standards and plant extracts were spiked with an internal standard

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(pentadecafluorooctanol; Pierce Chemicals, Rockford, IL) to enable correction for sample volumes and possible changes in Gas Chromatography-Mass Spectrometry (GC-MS) system

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responses. 2.6. Gas Chromatography-Mass Spectrometry Analysis

Separations were performed on an Agilent 7890B gas chromatograph coupled to an Agilent 5977A single quadrupole mass spectrometer. Splitless injections of 1 µL over 60 s, were made at 220°C onto a 30 m × 0.25 mm i.d. × 0.25 µm film, HP5-MS (Agilent) capillary column with a He flow of 1.2 mL min-1. The oven temperature program was 3 min at 35°C, 5°C min-1 to 70°C,

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and 10°C min-1 to 300°C, which was held for 5 min(Matich et al., 2015). Compounds were quantified by SIM (single ion monitoring); 2-phenylethylisothiocyanate (m/z 91), Sulforaphane nitrile (SFN,

m/z 82), N-methoxyindole-3- carboxaldehyde (m/z 175), Sulforaphane (SF, m/z

160), indol-3-acetonitrile (m/z 155), and indole-3-carboxaldehyde (m/z 144). The external

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standards, used for quantification, were SF (LKT Labs, St Paul Minn.) for SF and SFN, 2phenylethyl butanoate (synthesised in-house) for 2-phenylethylisothiocyanate, and methyl

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indole-3-carboxylate (Aldrich) for the indolic compounds. Identification of indolic compounds was based upon comparison of their fragmentation patterns with those in a NIST library (Nist Nist/Epa/Nih Mass spectral library and technology: Gaithersburg, 2011) and in other published identifications (Ernstsen and Sandberg, 1986; Sun et al., 2010). 2.7. Statistical Analysis

Statistical analysis was conducted using SAS 9.1 software. Nonlinear logistic function was fitted to evaluate the treatments effect on seed germination. ANOVA was used for comparing treatment groups. The Tukey’s test was conducted post hoc for comparing treatment group

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means at P ≤ 0.05 on three biological replicates. Appropriate data transformation was applied where the ANOVA assumptions were violated. 3. Results and discussion

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In this study broccoli sprouts were germinated and grown in several NaCl concentrations to determine how salt concentration affected their GL composition and total yield at 7 days. NaCl was also applied in combination with SA to test whether SA could alleviate the detrimental growth effects known to be caused by the salt stress (Ashraf and Harris, 2004) without

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suppressing the activation of the GL pathway. Transcripts encoding key enzymes in both aliphatic and indolic pathways were quantified to provide a more detailed regulatory view of the

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interaction between salt and SA on GL production.

3.1. Salt exposure reduces seed germination and growth of sprouts Broccoli seed germinated in the presence of increasing concentrations of NaCl had reduced and slower germination (Figure 1A). Without salt, 97% of the seed germinated and 50% of these did so by 24 h. When 80 mM NaCl was present, germination was still high at 95.5%, but the seed were slower to germinate, with 50% of the maximum germination percentage being at 40 h. The

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highest concentration of NaCl tested (160 mM) significantly lowered the germination percentage to 79% and the seed did not begin to germinate until 48 h, when by comparison seed in the control and 80 mM treatments had already fully germinated. Treatment of seed with 100 µM SA in the absence of salt did not affect the timing or percentage

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of seed that germinated compared with those in water controls (Figure 1B). The SA treatment also did not prevent the delayed germination of the seed caused by exposure to 80 mM NaCl.

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However, inclusion of SA with the 160 mM NaCl treatment led to more rapid seed germination (to about the same timing as observed for the 80 mM NaCl treatment) and prevented the loss in percentage germination.

Sprouts of seed germinated and held in 80 mM NaCl had greater fresh weight at day 7 than those in the 160 mM NaCl treatment, which did not show this increased fresh weight gain unless the treatment included SA (Figure 2A). The SA treatment also significantly reduced the decline in hypocotyl length caused by increased salt exposure (Figure 2B). There are different mechanisms by which SA could potentially improve seed germination and seedling growth under salinity stress. Lee et al. (2010) suggested that SA eliminates the salt

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inhibition of germination by lowering the amount of H2O2 produced by the salt treatment. They found that the antioxidants catechol and ascorbic acid, which also lowered the salt-induced accumulation of H2O2, similarly eliminated the salt inhibition of germination. Jayakannan et al. (2015), who studied the response of mutants defective in SA signalling or which constitutively

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expressed SA signalling, concluded that the SA-mediated tolerance was associated with limiting Na+ entry into root tissues and its subsequent long-distance transport in the shoot, and with preventing potassium loss.

3.2. Salt stress reduces the transcript abundance of key aliphatic and indolic pathway genes

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Continuous exposure of broccoli sprouts to increased concentrations of salt significantly decreased transcript abundance of CYP83B1 and appeared also to lower CYP79B2 transcript

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accumulation, although statistically the decline was not significant (Figure 3A&B). This suggested that the indolic GL pathway in broccoli sprouts was suppressed by salt stress. In addition, the transcript changes of the key aliphatic pathway genes Cyp83A1 and Cyp79F1 were also decreased by exposure to salt (Figure 3C&D). The lower expression of these genes was consistent with the trend for lower GL content of the salt-grown sprouts (Table 3). The SA treatment (in the absence of NaCl) increased Cyp79F1 transcript accumulation in the

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seven-day-old sprouts. This differs from other findings that have suggested the hormone affects the indolic but not the aliphatic pathway (Mikkelsen et al., 2003; Pérez-Balibrea et al., 2011). Although SA increased Cyp79F1 transcript accumulation in the absence of salt, it did not prevent the decline in Cyp79F1 transcript abundance caused by the salt exposure. In fact, the NaCl-

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mediated decline in transcript abundance of both aliphatic pathway genes was greater when SA was also included in the treatment (Figure 3A-D).

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Finding that SA inclusion prevented the NaCl treatments from inhibiting sprout growth, but did not prevent the decline in transcript abundance of key genes in both the aliphatic and indolic pathways, suggests that signals resulting directly from the growth inhibition response to NaCl exposure were not the cause of the transcriptional suppression of the GL biosynthetic pathway. Transcript abundance of three APSKs was examined to determine whether there was evidence at the transcriptional level for APSKs in the sulphur assimilation pathway being a control point for GL production. Transcript abundance of APSK1a and b both declined with increasing NaCl stress although only the decline in APSK1b was significant (Figure 4A&B). The transcript abundance of APSK2 also significantly declined in the sprouts treated with either 80 or 160 mM

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NaCl (Figure 4C). The decline in the APSK transcripts with salt stress correlated with the reduced GL seen in the tissues (Table 3). This confirms that the sulfur from the sulfur assimilation pathway is a regulatory point for GL accumulation (Mugford et al., 2009). The transcription factor AtMYB28 (HAG1; HIGH ALIPHATIC GL1) positively regulates

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aliphatic GL biosynthesis in Arabidopsis. When transiently expressed in leaves AtMYB28 activates promoter activity of MAM3, CYP79F1, and CYP83A1, and the gain-of-function AtMYB28 mutant of Arabidopsis has higher aliphatic GL content and higher transcript abundance of the aliphatic GL biosynthetic genes (Gigolashvili et al., 2007). However, in our study, the

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NaCl treatment did not significantly affect transcript abundance of BoMYB28 in the broccoli sprouts, although the transcript abundance did trend downwards (<2-fold) with increasing NaCl

BoMYB28 transcript accumulation.

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concentration (Figure 4D). Inclusion of SA with or without salt also had no significant effect on

Transcript abundance of myrosinase, which hydrolyses GR to SF, increased significantly in broccoli sprouts with increasing NaCl stress. The presence of SA, which alleviates the NaClinduced suppression of growth in the sprouts, did not prevent the NaCl mediated increase in transcript abundance of myrosinase (Figure 5A). ESM and ESP are both cofactors of myrosinase.

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ESM1 represses nitrile formation and favours production of isothiocyanates like SF, whereas ESP causes the formation of epithionitriles and nitriles such as sulforaphane nitrile, which has no anti-cancer activity (Matusheski and Jeffery, 2001; Zhang et al., 2006). NaCl stress of the sprouts

(Figure 5B&C).

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increased transcript abundance of ESM1, but had no effect on transcript abundance of ESP

To be confident that the SA and salt treatments were causing the responses in the broccoli

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sprouts, known SA- and salt-responsive transcripts were included in the transcription analysis (Figure 6). The PATHOGENESIS RELATED PROTEIN1 (PR-1) gene was chosen as a SA response control transcript because PR-1 transcripts have been shown to increase in response to SA (Jain et al., 2012; Van Loon et al., 2006). They are also induced in response to jasmonic acid and abiotic stress (Jain et al., 2012; Van Loon et al., 2006). P5CS, which encodes a delta-1pyrroline-5-carboxylate synthase that catalyses the rate-limiting enzyme in the biosynthesis of proline, was chosen because P5CS transcription is induced by NaCl (as well as abscisic acid) (Ben Rejeb et al., 2014; Liu and Zhu, 1997; Su et al., 2011). The transcript abundance of both

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genes was significantly increased by both salt and SA treatments compared with the control (Figure 6). 3.3. Glucosinolate concentration declines in broccoli sprouts grown in salt

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Broccoli GL profiles comprise aliphatic, indolic and aromatic GLs. Their relative proportions in broccoli and other Brassicaceae are tissue specific and can be further altered by various hormonal treatments (Avila et al., 2013; Bhandari et al., 2015). The nine major GLs identified in the seven-day-old sprouts in this study are indicated in Table 3. Five of the nine major GLs

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detected were of the aliphatic type. These in decreasing order of abundance were glucoraphanin (GR: 4-methylsulfinylbutyl), glucoerucin (GE: 4-methylthiobutyl), glucoiberverin (GI: 3methylthiopropy), gluconapin (GE: 3-butenyl), and glucoalyssin (GS: 5-methylsulfinylpentyl).

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Other studies have also reported that GR is the main GL in broccoli sprouts and immature floral heads (Alarcón-Flores et al., 2014; Baenas et al., 2014; Bellostas et al., 2007; Giambanelli et al., 2015; Pérez-Balibrea et al., 2010). The three indolic GL detected were derivatives of glucobrassicin. They were 4-hydroxyglucobrassicin (GB: 4-hydroxy-3-indolylmethyl), 4methoxyglucobrassicin (Mgb: 4-methoxy-3-indolylmethyl), and neoglucobrassicin (Ngb: 1methoxy-3-indolylmethyl). The one aromatic GL identified was gluconasturtiin (GN: 2-

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phenylethyl) and it was much lower in abundance than all other GLs except for glucoalyssin. The concentration of all nine GLs measured trended downwards in the seven-day-old sprouts as the salt treatment concentration was increased. This decline was significant for gluconapin, 4-

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methoxyglucobrassicin and neoglucobrassicin (Table 3). This correlated well with the decline in transcript abundance observed for all the GL biosynthetic pathway genes examined (Figures 3 & 4), suggesting that GL content of the sprouts is tightly controlled by transcriptional activity of the

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GL pathway as reported before by Baenas et al. ( 2014). These workers found that broccoli sprouts exposed to UVB for 24 h had both increased expression of CYP450 genes and higher GL content. Ku et al. (2013a) also reported a significant correlation between transcript abundance of the up-stream genes involved in the GL biosynthesis pathway and GL quantity. The effect of NaCl on GL profile and amount varies considerably depending on plant part, variety and growth conditions. López-Berenguer et al. (2008), who investigated the role of GLs in water relations and osmotic adjustment, found that leaves of 35-day-old broccoli plants that had been irrigated for the final 15 days with 80 mM NaCl had higher amounts of total GLs than

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controls held in Hoaglands media without salt. By contrast, Guo et al. (2013b) found no difference in total GLs compared with controls when broccoli sprouts were germinated in different concentrations of NaCl (80 and 100 mM NaCl). Another study showed that 40, 80 and 160 mM NaCl treatments did not affect the GR content of broccoli sprouts variety CL80, while

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the 160 mM NaCl treatment significantly enhanced the amount of GR in the variety ‘LangYan’(Guo et al., 2014). The differential effects of salt concentration on GL content were also seen in radish seedlings, where seven-day-old radish seedlings germinated and grown in 10

content than that in the control (Yuan et al., 2010).

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and 50 mM NaCl had lower total GL content, whereas those in 100 mM NaCl had a higher

In this study, the seven-day-old sprouts grown in SA (without salt) also had lower amounts of

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glucoalyssin and neoglucobrassicin than the sprouts held in water alone (Table 3). The combination of both NaCl and SA led to even lower amounts of gluconapin than in salt alone for the 80 mM NaCl treatment, but not for the 160 mM treatment. Other GLs did not change significantly. Previously, Pérez-Balibrea et al. (2011) showed that 100 µM SA did not change individual GLs significantly in broccoli sprouts, but they observed that total GLs increased. Similarly, Schreiner et al. (2011) found that turnips treated with 800 µM SA in hydroponic

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culture had higher amounts of total GL than controls. This resulted from elevated indolic and aromatic GL, and not aliphatic types, which actually declined.

3.4. Salt treatment induces sulforaphane production in broccoli sprouts

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In seven-day-old broccoli sprouts SF, which is the desirable hydrolysis product of GR because of its anti-cancer activity, is present in much lower amounts than the unwanted hydrolysis product SFN (300 vs. 10 000 ng g-1 FW)(Figure 7A vs B). However, treatment of the sprouts with 80

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mM NaCl and 160 mM NaCl increased the amount of SF to ~1500 and ~2000 ng g-1 FW respectively (Figure 7A), whereas 160 mM NaCl lowered SFN to approximately the same concentration (Figure 7B). Guo et al. (2014) also reported that 160 mM NaCl enhanced SF yield in three different varieties of broccoli sprouts. In our study, the increase in SF positively correlated with a salt-induced increase in transcript abundance of myrosinase (Figure 5A). Ku et al. (2013a) and Yang et al. (2015a & b) found that methyl jasmonate and zinc sulphate treatments of broccoli also increased BoMYO transcript abundance in the florets and sprouts, which likely led to the higher myrosinase activity they detected. We found that SF was

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preferentially made over SFN in the salt-treated sprouts, which was consistent with the increase in the ESM1 and not ESP transcript (Figure 5B&C). Increases in ESM1 to ESP favour the generation of isothiocyanates instead of nitrile forms (Hanschen et al., 2014; Lambrix et al., 2001), because the two co-factors act competitively to bind with myrosinase and when ESP

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protein is bound to the enzyme, myrosinase hydrolyses GLs to their nitrile forms (Matusheski et al., 2006). Co-treating the sprouts with SA did not affect the increase in SF caused by salt, but prevented salt from lowering the concentration of the nitrile form (Figure 7A&B).

The only other GL hydrolysis product measured in the broccoli sprouts that was present in

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increased amounts after the salt treatment was PEITC, which has antimicrobial activity (Chen et al., 2012) and induces apoptosis in many human cancer cells (Chou et al., 2015). It is an aromatic

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isothiocyanate derived from gluconasturtiin. The mode of action of PEITC has been reported to be similar to SA in that it induces hydrogen peroxide accumulation by inhibiting catalase activity (del Carmen Martínez-Ballesta et al., 2013). In our study, 160 mM NaCl increased PEITC and applying SA did not change this effect (Figure 7H). This is consistent with NaCl inducing high transcript abundance of BoMYO. A number of polyaromatic indolic compounds were detected in the broccoli sprouts (Figure 7). They are derived from indol-3-carbinol, which has been shown to

2005).

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suppress the proliferation of a wide variety of animal tumour cells (Aggarwal and Ichikawa, Indol-3-carbinol is produced endogenously from glucobrassicin or its derivatives

(Broadbent and Broadbent, 1998) and under acidic conditions converts into polyaromatic indolic compounds such as indole-3-carboxaldehyde, indole-3-acetonitrile, N- emethoxyindole-3-

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carboxaldehyde and N-methoxyindole-3-carbinol (Agerbirk et al., 1998). Treatment of the broccoli sprouts with the highest concentration of salt significantly decreased the three indolic

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compounds measured (Figure 7C-E). When 100 µM SA was included in combination with the 160 mM NaCl treatment, the reduction in the indolic compounds was less, and for indole-3carboxaldehyde there was no longer a reduction at all (Figure 7D). 4. Conclusions Our study revealed that SA treatment of sprouts prevented the negative growth effects caused by exposure of the sprouts to high concentrations of salt, but not the elevated SF content caused by the salt exposure (Figure 8). The combined treatment (160 mM NaCl/100 uM SA) either reduced or had no effect on transcript abundance of the GL biosynthetic pathway, which was reflected in either no change or a slight decrease in all GL metabolites measured. However, while GL

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content did not change substantially, this treatment increased transcript abundance of MYROSINASE and its cofactor BoESM1, whose encoded product directs MYROSINASE to produce the anti-cancer compound SF. This increased transcriptional abundance correlated well with the increased SF content of the sprouts, which was ~six times higher in the 160 mM

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NaCl/100 µM SA treatment than in the untreated controls. The prevention of the salt-mediated growth inhibition by SA further suggests that the salt-mediated transcriptional induction of the

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hydrolytic pathway was not indirectly caused by the negative effect of salt on sprout growth.

ACCEPTED MANUSCRIPT Contributions AE conceived and carried out the experimental work. AE and DH evaluated all of the experiments and wrote the manuscript. AS helped design experiments and analysed the data. AM helped process the samples for GL and isothiocyanate profiling and performed the LCMS determination. DH, MB and MM supervised the research. All authors read and

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approved the final manuscript. Acknowledgments

We thank PFR staff Ronan Chen for technical assistance and Duncan Hedderley for help with the statistics in Table 3. This work was supported by Plant & Food Research CORE funding

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and by an Iranian Government Ministry of Science, Research and Technology travel award to

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ACCEPTED MANUSCRIPT References

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Figures

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Figure 1. Fitted curves of logistic function on the effect of sodium chloride (NaCl) and salicylic acid (SA) treatments on timing of broccoli seed germination and percentage germination. (A) seed germination in 0, 80 and 160 mM NaCl without SA. (B) seed germination in 0, 80 and 160 mM NaCl with 100 µM SA. Each data point is the mean of three biological replicates, which were measured at three time points during the day (0900, 1300, and 1600 h). Each replicate consisted of 60 sown seeds.

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Figure 2: Fresh weight (A) and hypocotyl length (B) of seven-day-old broccoli sprouts that were germinated and grown in sodium chloride (NaCl) and salicylic acid (SA) treatments. Each data column is the mean ±SE of three biological replicates with each replicate

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consisting of twenty seedlings. Different letters indicate significant differences at P < 0.05.

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(A)

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Figure 3. Effect of salt (NaCl) and salicylic acid (SA) treatments on transcript abundance of genes in the indolic and aliphatic GL pathways in broccoli sprouts. Sprouts were germinated and held in treatments for 7 days and transcript abundance of genes BoCYP83B1 (A) and BoCYP79B2 (B) in the indolic pathway and BoCYP83A1 (C) and BoCYP79F1 (D) in the aliphatic pathway was determined by quantitative PCR. Transcript abundance changes were normalised to ACTIN. Each data column is the mean ±SE of three biological replicates assayed in triplicate. Different letters indicate significant differences at P < 0.05.

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Figure 4. Effect of salt (NaCl) and salicylic acid (SA) treatments on transcript abundance of BoAPSK1 and BoAPSK2, BoMYB28 in broccoli sprouts. Sprouts were germinated and held in treatments for 7 days. Transcript abundance changes were determined by quantitative PCR and normalised to ACTIN. Each data column is the mean ±SE of three biological replicates assayed in triplicate. Different letters indicate significant differences at P < 0.05.

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Figure 5. Effect of salt (NaCl) and salicylic acid (SA) treatments on transcript abundance of GL hydrolytic genes relative to untreated control in broccoli sprouts. (A) BoMYO (B) BoESP, (C) BoESM1, Sprouts were germinated and held in treatments for 7 days. Transcript abundance changes were determined by quantitative PCR and normalised to ACTIN. Each data column is the mean ±SE of three biological replicates assayed in triplicate. Different letters indicate significant differences at P < 0.05.

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Figure 6. Effect of salt (NaCl) and salicylic acid (SA) treatments on transcript abundance of (A) salt-inducible delta-1-pyrroline-5-carboxylate synthase A-like (BoP5CS) and (B) salicylic acid-inducible pathogenesis-related (BoPR1) genes. Transcript abundance changes were determined by quantitative PCR and normalised to ACTIN. Each data column is the mean ±SE of three biological replicates assayed in triplicate. Different letters indicate significant differences at P < 0.05.

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(F)

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2-phenylethylisothiocya nate (ng.g-1 FW)

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SA=0µM SA=100µM

A AB

250 200 150

B

AB

B B

100 50 0 NaCl =0mM

NaCl =80mM

NaCl =160mM

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Figure 7. Effect of salt (NaCl) and salicylic acid (SA) treatments on GL hydrolysis products of broccoli sprouts 7 days after treatments. Each data column is the mean ±SE of 3 biological replicates. Different letters indicate significant differences at P < 0.05.

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Figure 8. Effect of 160 mM sodium chloride and 100 µM salicylic acid treatment on GL biosynthesis and hydrolysis pathways in broccoli sprouts. SF: sulforaphane, PEITC: 2phenylethylisothiocyanate.

ACCEPTED MANUSCRIPT Table 1. Primers used in quantitative Real Time Polymerase Chain Reaction. Size Gene Primer (5´-3´) Tm Description Pathway (bp)

BoCYP79F1

cytochrome P450 (CYP79F1)a

BoCYP83A1

cytochrome P450 (CYP83A1)a

BoMYO

myrosinase (MYO)a

epithiospecifier (ESP) proteina

BoESP

BoESM1

epithiospecifier modifier (ESM)a

BoAPK1a

indolic

aliphatic

aliphatic

hydrolysis

hydrolysis

hydrolysis

synthesis

F

ACGAGATAAACCGGAGATCG

59.2

R

AGCCAAGTCCTTCTCAGTCG

59.6

F

CTCTCTTGAGACGCGCACTA

59.8

R

ACGGAACCGAGATGAAGAGA

59.5

F

TCCGATGGTTCTCATGTTGA

R

AACCGGATATCGCATGTTTC

F

CAAGTGGTTCATCCCCATCT

R

TCAAGACGCAAGACGTCAAC

F

TCACCTTTCCACCAAATTCC

59.3

R

AACGCCTTTCGTTACCCTCT

55.8

F

CTTGGACGGAGAGATTGACC

57.8

R

CGAGAAGCTCACATGGCATA

57.7

F

CCGGAGCCCCAAGAATAGAA

61.0

R

ATTCCAAACGGAATCCCGCC

59.2

F

AACTTCACCGATCGAAATGG

57.8

R

TGTGTGCGGTTGAAAAGGTA

57.7

F

GGATTGATGACCCTTACGA

59.6

R

GAAGGTAACCCGCAGTCT

60.0

F

CTGGAATCGACGACCCTTAC

59.7

R

GTTTTCAGCCATCTGACGTG

59.9

F

CCACCATTGTTACACCTTGCT

57.3

R

AACCTTTGGGTCAACGAGAA

58.8

F

CGAGCAGTAGAATCCGTGATAG

58.0

R

GCTATGCAGCTGAGAACATAGA

58.2

F

CCCAAGAGAAAAGGTTTCAA

57.7

R

CCCTAAACTTGGGACTAACAACC

58.5

F

TCTCGATGGAGGAGCTGGTT

55.8

R

GATCCTTACCGAGAGAGGTT

60.3

F

GAGACTTGTGTGAGGGAGAAA

59.9

R

CCAACGGTATGAAATGCTCAAC

60.1

APSkinase

synthesis APSkinase

BoAPSK2

APSkinase pathogenesisrelated (PR) protein

synthesis

SAinducible

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BoPR

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BoAPK1b

delta 1pyrroline-5carboxylate synthetase

NaClinducible

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BoP5CS

BoMYB28

Transcription factor Mybrelated protein 28

BoACT1

actin (ACT1)

BolPP2AA2

protein phosphatase 2A subunit A2, At3g25800 ortholog

aliphatic

reference

reference

139

138

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BoCYP83B1

cytochrome P450 (CYP83B1)a

indolic

59.8

140

60.1 60.0

140

59.8

SC

cytochrome P450a

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BoCYP79B2

Bo, Brassica oleraceae; a Ref:(Ku et al., 2013a).

140

140

97

111

161

101

140

106

139

140

140

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SFN 10072±1487 12013±1077 6424±1591

MIC 18±7 99±7 3±1

SF 1196±45 3533±540 67±12

I3A 1799±166 2880±161 1661±353

I3C 175±3 598±142 46±2

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PEITC: 2-phenyl-ethylisothiocyanate, SFN: sulforaphane nitrile, MIC: N-methoxyindole-3-carboxaldehyde, SF: sulforaphane, I3A: indole-3-acetonitrile, I3C: indole-3-carboxaldehyde.

ACCEPTED MANUSCRIPT Table 3. Effect of sodium chloride and salicylic acid treatments on GL content of 7-day-old broccoli sprouts. Data are mean of 3 biological replicates. Concentrations determined by LCMS against glucoerucin for the aliphatic and phenolic glucosinolates, and against glucobrassicin for the indolic glucosinolates. SA

GLs ( µg.g-1 FW) GR

A

A

GS

GEA

(mM)

(µM)

0

0

0

100

429

2.7

18.4

80

0

648

3.8

18.8

80

100

468

2.5

13.9

160

0

552

3.5

160

100

561

SA (1 and 12 df)

4.5

27.8

565

GIA

GBI

NgbI

GNAR

total

328

9.8

153

236

6.3

1357.50

193

285

7.9

1906.19

147

192

6.7

1414.84

138

114

173

7.2

1507.69

188

134

184

10

1707.43

0.662

0.122

0.082

0.621

0.127

0.052

0.053

0.666

0.916

0.190

0.322

0.124

0.205

142

166

393

94

107

589

121

143

455

95

111

14.7

472

94

3.6

21.5

584

114

0.941

0.051

0.282

0.379

0.240

Salt (2 and 12 df)

0.950

0.698

0.069

0.830

0.723

0.183

SA.Salt (2 and 12 df)

0.280

0.238

0.030

0.244

0.193

0.099

226

M AN U

ANOVA p values

MgbI

SC

734

GP

A

RI PT

NaCl

2068.37

AC C

EP

TE D

GR: Glucoraphanin, GS: Glucoalyssin, GP: Gluconapin, GE: Glucoerucin, GI: Glucoiberverin, GB: 4Hydroxyglucobrassicin, Mgb: 4-Methoxyglucobrassicin, Ngb: Neoglucobrassicin, GN: Gluconasturtiin, A: aliphatic, I: indolic, AR:aromatic.

ACCEPTED MANUSCRIPT

Highlights

EP

TE D

M AN U

SC

RI PT

NaCl treated broccoli sprouts had more sulforaphane (SF) content but impaired growth Salicylic acid (SA) co-treatment improved growth without compromising SF increase MYROSINASE and EPITHIOSPECIFIER MODIFIER1 transcript abundance was also increased SF content increased 6-fold in sprouts treated with 160 mM NaCl/100 uM SA

AC C

• • • •

ACCEPTED MANUSCRIPT Contributions

AC C

EP

TE D

M AN U

SC

RI PT

AE conceived and carried out the experimental work. AE and DH evaluated all of the experiments and wrote the manuscript. AS helped design experiments and analysed the data. AM helped process the samples for GL and isothiocyanate profiling and performed the LCMS determination. DH, MB and MM supervised the research. All authors read and approved the final manuscript.